Interleukin‐7 supports survival of T‐cell receptor‐β‐expressing CD4− CD8− double‐negative thymocytes

Among the milestones that occur during T-cell development in the thymus is the expression of T-cell receptor-β (TCR-β) and the formation of the pre-TCR complex. Signals emanating from the pre-TCR trigger survival, proliferation and differentiation of T-cell precursors. Although the pre-TCR is essential for these cell outcomes, other receptors, such as Notch and CXCR4, also contribute. Whether interleukin-7 (IL-7) participates in promoting the survival or proliferation of pre-TCR-expressing cells is controversial. We used in vitro and in vivo models of T-cell development to examine the function of IL-7 in TCR-β-expressing thymocytes. Culturing TCR-β-expressing CD4⁻ CD8⁻ double-negative thymocytes in an in vitro model of T-cell development revealed that IL-7 reduced the frequency of CD4⁺ CD8⁺ double-positive thymocytes at the time of harvest. The mechanism for this change in the percentage of double-positive cells was that IL-7 promoted the survival of thymocytes that had not yet differentiated. By preserving the double-negative population, IL-7 reduced the frequency of double-positive thymocytes. Interleukin-7 was not required for proliferation in the in vitro system. To follow this observation, we examined mice lacking CD127 (IL-7Rα). In addition to the known effect of CD127 deficiency on T-cell development before TCR-β expression, CD127 deficiency also impaired the development of TCR-β-expressing double-negative thymocytes. Specifically, we found that Bcl-2 expression and cell cycle progression were reduced in TCR-β-expressing double-negative thymocytes in mice lacking CD127. We conclude that IL-7 continues to function after TCR-β is expressed by promoting the survival of TCR-β-expressing double-negative thymocytes.     

Interleukin-7 inhibits pre-T-cell differentiation induced by the pre-T-cell receptor signal and the effect is mimicked by hGM-CSF in hGM-CSF receptor transgenic mice

We have previously reported that human granulocyte-macrophage colony-stimulating factor (hGM-CSF) causes a stage-specific inhibition of T-cell receptor (TCR) alphabeta cell development in the thymus of transgenic mice constitutively expressing the hGM-CSF receptor. Since it has been reported that the addition of interleukin-7 (IL-7) to fetal thymic organ culture (FTOC) has similar effects, we compared the effects of IL-7 and hGM-CSF on TCR(alphabeta) cell development in hGM-CSF receptor transgenic mice. We reconstituted fetal lobes with sorted pre-T, or post pre-T CD4(-)CD8(-) precursor cells. The addition of either IL-7 or hGM-CSF to these cultures suppressed further differentiation of pre-T cells but not post pre-T cells. At the same time, the cell number was increased, suggesting that pre-T-cell proliferation is stimulated by these cytokines. Furthermore, the differentiation of recombination-activating gene-1 (RAG-1)-deficient pre-T cells in response to anti-CD3 antibody stimulation was suppressed by either IL-7 or hGM-CSF, suggesting that these cytokines inhibit the pre-T-cell receptor (pre-TCR) signal. This inhibition is unexpected because the pre-TCR signal and the IL-7 signal have previously been considered to be co-operative. Recent analysis of the downstream events of IL-7 receptor and GM-CSF receptor revealed that they share common signal transduction molecules. Our results show that IL-7 is able to promote pre-T cell proliferation and to suppress differentiation induced by the pre-TCR signal. GM-CSF can mimic these biological activities of IL-7 when the pre-T cells express GM-CSF receptors. Our data suggest that both timing and level of activation of the IL-7 signalling pathway must be precisely regulated to facilitate the differentiation of thymocytes.     

A nonredundant role for the adapter protein Shc in thymic T cell development

Signaling via the pre-T cell receptor (pre-TCR) regulates survival, proliferation, allelic exclusion and differentiation of thymocytes. The role played by the adapter protein Shc in T cells has remained controversial, and its role in pre-TCR signaling has not been addressed. We examined Shc function in thymic T cell development using two genetic approaches. Cre-loxP-mediated inducible expression in transgenic mice of a phosphorylation-defective mutant of Shc impaired signaling through the pre-TCR as well as subsequent proliferation and differentiation. Conditional deletion of the Shc locus in thymocytes also affected thymic maturation at the same pre-TCR developmental stage. Thus, both Shc expression and its tyrosine phosphorylation play an essential and nonredundant role in thymic T cell development.     

Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation

ABSTRACT: BACKGROUND: The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system. RESULTS: In this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRalphabeta+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vbeta repertoire. CONCLUSIONS: Our study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.     

Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.     

Role of interleukin-7 in T-cell development from hematopoietic stem cells

Summary: All lymphocytes are derived from hematopoietic stem cells (HSC). The interleukin-7 receptor (IL-7R) transduces non-redundant signals for both T and B-cell development from HSC. The upregulation of the IL-7R occurs at the stage of the clonogenic common lymphoid progenitor, a recently identified population that can give rise to all lymphoid lineages (T, B and natural killer cells) at a single cell level. The IL-7R plays a critical role in the rearrangement of immunoglobulin heavy chain genes required for B-cell development, IL-7R expression is critically regulated in developing thymocytes; thytnocytes that fail the positive selection process down-regulate the IL-7R, but those undergoing positive selection upregulate or maintain IL-7R expression. Recent data indicate that IL-7 signaling enhances the survival of developing thymocytes and mature T ceils, presumably by its upregulating Bcl-2. Detailed analysis of the signaling cascades activated by the IL-7R may help to reveal the differential roles of IL-7 signaling in T and B-cell development.     

Intrathymic IL-7: the where, when, and why of IL-7 signaling during T cell development

The thymus is the birthplace of all T lineage cells. But the thymus is also a cradle as it provides the environment for further maturation and differentiation of immature thymocytes. While many factors contribute to make the thymus a unique place for T cell development, here we review the essential role of intrathymic interleukin-7 (IL-7). In the absence of IL-7 signaling, survival, proliferation and differentiation of immature thymocytes are all severely impaired. Consequently, IL-7 is critical to nurture and guide T precursor cells through the diverse steps of thymic maturation. Interestingly, even as IL-7 signaling is such a critical factor, IL-7 signaling must be also actively suppressed during specific stages of T cell differentiation. These contradictory observations are puzzling but can be satisfactorily explained when understanding the developmental context of IL-7 signaling. In this regard, here we will discuss the spatiotemporal expression of intrathymic IL-7 and address the stage-specific effects of IL-7 signaling in developing thymocytes. Specifically, we will review other facets of intrathymic IL-7 beyond its role as a pro-survival factor and so clarify and reaffirm the unique role of IL-7 as a prime factor in T cell development and differentiation.     

IL-21 promotes T lymphocyte survival by activating the phosphatidylinositol-3 kinase signaling cascade

IL-21 is a Type I cytokine, which uses the common gamma chain (gamma(c)) in its receptor. As members of the gamma(c) cytokine/cytokine receptors family play crucial role in the differentiation, activation, and survival of lymphocytes, we have investigated if IL-21 could promote T cell survival and thus, contribute to T cell homeostasis and expansion. Unlike most gamma(c) cytokine receptors, we report that IL-21R is constitutively expressed by all mature T lymphocytes and that stromal cells of lymphoid organs are a constitutive source of IL-21. These observations are reminiscent of what is observed for IL-7/IL-7R, which control T cell survival and homeostasis and suggest a role for IL-21 in T cell homeostasis. Indeed, our results show that IL-21 is a survival factor for resting and activated T cells. Moreover, the ability of IL-21 to costimulate T cell proliferation is mediated by enhancing T cell viability. Further investigation of how IL-21R signaling induces T cell survival shows for the first time that IL-21 binding to its receptor activates the PI-3K signaling pathway and induces Bcl-2 expression. Moreover, the activation of the PI-3K signaling pathway is essential for IL-21-mediated T cell survival. Our data provide a new role for IL-21 in the immune system, which might be used to improve T cell homeostasis in immunocompromised patients.     

Establishment of the major compatibility complex-dependent development of CD4+ and CD8+ T cells by the Cbl family proteins

Casitas B cell lymphoma (Cbl) proteins are negative regulators for T cell antigen receptor (TCR) signaling. Their role in thymocyte development remains unclear. Here we show that simultaneous inactivation of c-Cbl and Cbl-b in thymocytes enhanced thymic negative selection and altered the ratio of CD4(+) and CD8(+) T cells. Strikingly, the mutant thymocytes developed into CD4(+)- and CD8(+)-lineage T cells independent of the major histocompatibility complex (MHC), indicating that the CD4(+)- and CD8(+)-lineage development programs are constitutively active in the absence of c-Cbl and Cbl-b. The mutant double-positive (DP) thymocytes exhibited spontaneous hyperactivation of nuclear factor-kappa B (NF-kappaB). Additionally, they failed to downregulate the pre-TCR and pre-TCR signaling. Thus, our data indicate that Cbl proteins play a critical role in establishing the MHC-dependent CD4(+) and CD8(+) T cell development programs. They likely do so by suppressing MHC-independent NF-kappaB activation, possibly through downmodulating pre-TCR signaling in DP thymocytes.     

Expression of cytokines and their receptors by human thymocytes and thymic stromal cells.

The repertoire of cytokine and cytokine receptor mRNA expressed by unstimulated human thymocytes and thymic stromal cells was explored by a quantitative polymerase chain reaction (PCR) using sequence specific internal standards. Of the 18 cytokines tested we found a considerable overlap in the expression of cytokines by human thymocytes and by thymic stromal cells; both cell types express the mRNA for interleukin-1 beta(IL-1, IL-6, IL-7 and tumour necrosis factor-alpha (TNF-alpha). However, there are substantial differences in the levels of cytokine mRNA expressed in these two types of cells as revealed by the quantitative PCR assay. Stromal cells express considerably higher levels of IL-1 beta and IL-6 than thymocytes (14- and 27-fold respectively). In addition, a number of cytokines such as lymphotoxin and interferon-gamma (IFN-gamma), are expressed exclusively in thymocytes whereas others such as stem cell factor (SCF), IL-1 receptor antagonist-2 (IRAP-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are produced only in stromal cells. There is a complete overlap in the expression of a group of cytokine receptors tested in thymocytes and thymic stromal cells; these include IL-1R, IL-2R, IL-6R, IL-7R, TNFR and stem cell growth factor receptor (c-KIT). The expression of specific cytokines by thymic stromal cells and the parallel expression of their receptors on thymocytes under physiological conditions, support the hypothesis that these cytokines participate in paracrine interactions between these two cell populations during thymocyte differentiation.     

Critical roles of the PI3K/Akt signaling pathway in T cell development

Thymocyte development requires an integration of extracellular cues to enforce lineage commitment at multiple defined checkpoints in a stage-specific manner. Critical signals from the pre-TCR, Notch, and the receptor for interleukin-7 (IL-7) dictate cellular differentiation from the CD4(-)CD8(-) (double negative) stage to the CD4+CD8+ (double positive) stage. The PI3K/Akt signaling pathway is required to translate these extracellular signaling events into multiple functional outcomes including cellular survival, proliferation, differentiation, and allelic exclusion at the beta-selection checkpoint. However, a complete understanding of the contributions made by the PI3K/Akt pathway in thymocyte development has not been straightforward. This review highlights studies that support the model that the PI3K/Akt pathway is essential for thymocyte survival. We provide new evidence that Akt-mediated survival is not solely due to the increased expression of Bcl-xL but also is a consequence of the role played by Akt to support metabolism in proliferating thymocytes.     

Hedgehog signaling regulates differentiation from double-negative to double-positive thymocyte

The hedgehog (Hh) signaling pathway is involved in the development of many tissues. Here we show that sonic hedgehog (Shh) is involved in thymocyte development. Our data suggest that termination of Hh signaling is necessary for differentiation from CD4-CD8-double-negative (DN) to CD4+CD8+ double-positive (DP) thymocyte. Shh is produced by the thymic stroma, and Patched and Smoothened (Smo), the transmembrane receptors for Shh, are expressed in DN thymocytes. A neutralizing monoclonal antibody against Shh increases differentiation of DN to DP thymocytes, and Shh protein arrests thymocyte differentiation at the CD25+ DN stage, after T cell receptor beta (TCRbeta) gene rearrangement. We show that one consequence of pre-TCR signaling is downregulation of Smo, allowing DN thymocytes to proliferate and differentiate.     

On the brink of becoming a T cell

Recent studies provide fresh insight into the mechanisms by which precursor cells are committed to and develop within the T-lymphocyte lineage. Precursor/product studies have identified developmental stages between that of the pluripotent hematopoietic stem cell and thymocytes committed to the T lineage. Specific ligands and signaling pathways interacting with the Notch-1 receptor and its ability to influence commitment within the lymphoid lineage have been described. Although the structural features or putative ligands endowing the pre-TCR with constitutive signaling capacity remain elusive, numerous distal mediators of pre-TCR signaling have been identified. It remains for the future to determine what roles they may have in survival, proliferation, lineage commitment and allelic exclusion of TCR genes. Receptor editing and lineage commitment of alphabeta T cells still represent controversial topics that need further study.     

Regulation of thymocyte differentiation: pre-TCR signals and beta-selection

The specificity of the adaptive immune response is, in part, dependent on the clonal expression of the mature T cell receptor (TCR) on T lymphocytes. One mechanism regulating the clonality of the TCR occurs at the level of TCR-beta gene rearrangements during lymphocyte development. Expression of a nascent TCR-beta chain together with pre-Talpha (pTalpha) and CD3 molecules to form the pre-TCR complex, represents a critical checkpoint in T cell differentiation known as beta-selection. Indeed, failure to generate a functionally rearranged TCR-beta chain at this stage of development results in apoptosis. Signals derived from the pre-TCR complex trigger a maturation program within developing thymocytes that includes: rescue from apoptosis; inhibition of further DNA recombination at the TCR-beta gene locus (allowing for the clonality of antigen receptor expression; allelic exclusion); and induction of proliferation and differentiation. The signaling mechanisms that control this developmental program remain largely undefined. Here, we discuss recent evidence investigating the molecular mechanisms that regulate thymocyte differentiation downstream of pre-TCR formation.     

Distinct roles of the phosphatidylinositol 3-kinase and STAT5 pathways in IL-7-mediated development of human thymocyte precursors.

Here, we define the IL-7R-activated signal that promotes survival and proliferation of T cell progenitors and demonstrate that it is distinct from the signals that induce differentiation. We show that IL-7 activates PKB and STAT5 in human thymocytes. Into T cell precursors we introduced chimeric receptors with a cytoplasmic domain of the IL-7R that is no longer able to activate PI-3K/PKB and STAT5 and tested the transduced cells in a fetal thymic organ culture. We also examined the T cell precursor activity of progenitors expressing dominant-negative forms of PI-3K or STAT5B. These experiments revealed that PI-3K/PKB activation is essential for the survival and proliferation of T cell precursors and suggest that STAT5 activated by IL-7 mediates T cell differentiation.